1. Field of the Invention
The present invention relates to the removal of heparin from blood plasma test samples.
2. Description of the Prior Art
Heparin is widely used as a parenteral anticoagulant for the treatment of thromboembolic patients, for the prophylactic treatment of high risk embolic patients and to reduce the incidence of deep-vein thrombosis after major surgery. Thromboembolic conditions are characteristic of disease states such as coronary thrombosis, venous thrombosis, pulmonary embolism and the like. During treatment, it is necessary to determine the patient's clotting time in the absence of heparin, i.e., the true blood plasma clotting time. Thus, it is necessary to neutralize or remove the heparin from the sample of the patient's blood plasma prior to coagulation testing.
Conventionally, heparin is neutralized by reaction with protamine, as described by Allen, J. G., et al., "A Protamine Titration as an Indication of a Clotting Defect in Certain Hemorrhagic States", in J. Lab. Clin. Med. 34:473-476 (1949) and by Perkins, H. A., "Neutralization of Heparin In Vitro with Protamine; A Simple Method of Estimating the Required Dose", J. Lab. Clin. Med. 48:223-226 (August, 1956). Polybrene, a synthetic polymerized quaternary ammonium salt, has also been used to neutralize heparin (see Godal, H. C., "A Comparison of Two Heparin Neutralizing Agents: Protamine and Polybrene", Scandinav. J. Clin. & Lab. Invest. 12:466-457, (1960).
While the above-mentioned procedures have been used successfully, a tedious titration is required since protamine and polybrene are soluble in plasma. Any excess not combined with heparin will remain in the plasma, frequently undetected, and interfere with the coagulation test. Thus, the accuracy of the coagulation test results are questionable. The fact that the titration method for neutralization of heparin is of limited accuracy has been recognized in the literature: differences of as much as 5 to 10 mcg. of heparin per milliliter of blood have been reported by Wright, J. S., et al., "Heparin Levels During and After Hypothermic Perfusion", in J. Cardiovas. Surg. 5:244-250 (1964).
In order to overcome the above-mentioned deficiencies, Thompson, et al. (J. Lab. Clin. Med. 88:922-929, December, 1976) developed a chromatographic technique for removing heparin and protamine from plasma samples. Columns of ECTEOLA-cellulose (a moderately basic anion exchange resin) were used to adsorb up to 300 U. of heparin from 1 milliliter of plasma; and columns of carboxymethyl cellulose (a cation exchange resin) were used to adsorb protamine. However, the preparation and use of ion exchange columns prior to testing plasma for clotting time is time consuming, cumbersome and inconvenient. Additionally, at least a milliliter of plasma sample is taken up by the ion exchange columns and is unavailable for further testing. This is a serious disadvantage in pediatric cases where only small amounts of plasma sample can be obtained for testing.
In view of the deficiencies of the conventional art methods, there is a need for a simple, rapid procedure for removal of heparin from blood plasma samples without adversely affecting subsequent coagulation testing of the plasma sample.
Co-pending application Ser. No. 931,033, filed Aug. 4, 1978 by Arthur L. Babson and James E. Turner, entitled REMOVAL OF HEPARIN FROM BLOOD PLASMA SAMPLES USING AN INSOLUBLE PROTAMINE REACTION PRODUCT, describes a method for removing heparin using an insoluble protamine/glutaraldehyde reaction product; and co-pending application Ser. No. 931,031 filed Aug. 4, 1978 by James E. Turner, James R. Butler and Arthur L. Babson, entitled REMOVAL OF HEPARIN FROM BLOOD PLASMA SAMPLES USING TRIETHYLAMINOETHYL CELLULOSE describes a method for removing heparin using fibrous triethylaminoethyl cellulose.